Process of causing lysis of blood clots,and composition



Dec. 22, 1970 A. P. TRUANT ETAL 3,549,748

PROCESS OF CAUSING LYSIS OF BLOOD CLOTS, AND COMPOSITION Original Filed Feb. 20, 1959 5mn www" 3,549,748 PROCESS OF CAUSING LYSIS OF BLOOD CLOTS, AND COMPOSITION Aldo P. Truant and Fritz G. Nordstrom, Worcester, Mass., assignors to Astra Pharmaceutical Products, Inc., Worcester, Mass., a corporation of New York, and The Governors of The University of Toronto, Toronto, Ontario, Canada Original application Feb. 20, 1959, Ser. No. 794,729, now Patent No. 3,256,157. Divided and this application June 13, 1966, Ser. No. 557,155

Int. Cl. A61k 17/00, 19/00 U.S. Cl. 424-94 6 Claims ABSTRACT OF THE DISCLOSURE Fibrinolytic agents derived from specific mold cultures are intravenously administered for causing lysis of blood clots in veins and arteries of humans and warm-'blooded animals and are topically applied to purulent and necrotic Wounds to accelerate healing. The injectable solutions may contain heparin and/or plasmin. The topically applicable preparations are ointments, powders and the like. They may contain an antibiotic.

The present invention is a division of copending application Ser. No. 794,729, now Pat. No. 3,256,157 led Feb. 20, 1-959 and entitled Agents Having a Fibrinolytic Activity and Being Derived From Molds, and a Process of Making and Using Same.

The present invention relates to a method of causing lysis of blood clots and to composition useful therefor. As is known, the clotting of blood will occur in the intact vascular system. If a clot does develop, the problem of thrombosis may arise and the resulting thrombus may lead to infarction and, as a result thereof, to necrosis of the tissue supplied by the affected blood vessel. A blood clot or embolus forced by the blood current from a thrombosed vessel into a smaller one or formed in a coronary artery may cause obstruction of vital areas of the body and often death.

A number of agents have been used in therapy to prevent an extension of the blood clot or to cause its dissolution. Heparin or other anticoagulants, for instance, have been administered for their property of prolonging the clotting time of blood which facilitates clot resolution and prevents extension of an existing intravascular clot.

Other agents, such as the enzyme streptokinase have an activating effect on the precursor of the brinolytic enzyme probrinolysin which causes cleavage of brin, i.e. the material comprising the blood clot, into soluble polypeptides. An agent of similar activity is streptodornase. The enzyme trypsin has also been used for the lysis of brin and plasma clots, likewise the enzyme plasmin obtained from human blood.

These known agents, however, have a number of disadvantages.

1) Heparin and coumarins do not directly affect an existing blood clot and they do not limit the damage to the affected vascular tissue.

(2) Trypsin, in doses insutiicient to induce clot lysis, causes an acceleration of the clotting mechanism and in- United States Patent O 3,549,748 Patented Dec. 22, 1970 ICC travascular coagulation. There is no satisfactory means available to determine a priori the dose necessary for brinolytic activity.

(3) Streptokinase, although not antigenic, possesses a pyrogenic action.

(4) Plasmin is expensive and has an antigenic action.

(5) Both trypsin and streptokinase measurably affect other clotting and plasma constituents. Therefore, these known agents have found only limited application in therapy.

`It is one object of the present invention to provide a simple and eifective method of causing lysis of blood clots Without substantially affecting the other blood and plasma constituents.

Another object of the present invention is to provide a pharmaceutical composition useful in carrying out said method of causing lysis of blood clots.

Other objects of the present invention and advantageous features thereof will become apparent as the description proceeds.

In principle the present invention consists in administering to humans and warm-blooded animals a composition containing as `blood clot lysing agent, the ibrinolytic agent isolated from cultures of specilic molds that are capable of producing such agents.

The preparation and isolation of such blood clot lysing agent has been described in detail in said copending application Ser. No. 794,729, now Pat. No. 3,256,157 which is indicated by reference herein.

As stated therein, blood clot lysing agent can 'be isolated and produced from any strain or species of mold which, when tested by the test methods given therein, shows selective brinolytic activity. While the methods of isolating and purifying the brinolytic agent are described in great detail in said Pat. No. 3,256,157, it is understood that other methods may be used provided they yield iibrnolytic agents which exhibit the desired fibrinolytic activity in said tests.

Especially active brinolytic agents acording to the present invention are those products which are obtained by the tannic acid precipitation method as described in detail in said Pat. No. 3,256,157.

The unit of activity of the iibrinolytic agent is expressed as the amount of activity contained in one mg. of dry material which causes fragmentation of the brin plate prepared as described in said Pat. No. 3,256,157, to a diameter of 1 cm. after 60 minutes at 37 C., when applied to such a plate in an amount of 0.1 cc. of an 0.1% aqueous solution. For instance, the tannic acid precipitates correspond to 2 to 4 units/mg..

More particularly two strains of the species Aspergillus oryzae, which are available to the public from the Quartermaster Research and Engineeringl Center, United States Army, at Natick, Mass., under the depository numbers B-82i and B-1273, are capable of producing especially large amounts of the new fibrinolytic agents. One strain of the species Aspergillus JZa'vus, also available to the public from said Quartermaster Research and Engineering Center under the depository number B-4m, and one strain of the genus Absidia, species Absdia coerulea, also available to the public from said Quartermaster Research and Engineering Center under the depository number D-lOl, have also proved to produce said new brinolytic Neopeptone 10 Bacto-dextrose 40 Bacto-agar 15 Water to 1000 cc.

After cultivating the molds on said solid medium at SO-37 C. for five to ten days, the resulting inoculum was transferred to a liquid medium prepared from Sucrose 7.2

Dextrose 3.6 MgSO4 (crystals) 1.23 KHgPO.,t 13.69 KNO3 2.0

And H2O to 1000 cc.

Aliquots of 100 cc. were transferred to Erlenmeyers flasks of 250 cc. capacity. A sample of the Sabouraud culture, approximately l cm. in diameter, was transferred to each flask. Cultures were incubated at 29-30 C.

After a specific interval of time, the content of the flasks was filtered on Whatman No. 2 paper under sterile conditions. To one volume of the clear filtrate, occasionally stained by pigments produced by the culture, there were added two volumes of 95% alcohol precooled at -20 C. A precipitate formed almost immediately and separated slowly to the bottom. After standing overnight at 20 C., tubes containing the mixture were centrifuged at 2,000 r.p.m./l5 min./ 4 C. and the supernatant liquid was discarded.

The precipitate is then dried in a vacuum at 37 C. According to the activity of the precipitate amounts between 0.5 Ing. and 10.0 mg. of the dried material are suspended in 1 cc. each of distilled Water, saline solution, or phosphate buffer solution of a pH of 5.5, the total volume of the suspension being one tenth of the volume of the original filtrate.

Culture media containing a substantial amount of .phosphates have a favorable effect on the amount of fibrinolytic agent produced by the fungus. It is. of course, understood that the culture medium contains a source of carbon, such as starches, dextrin, dextrose, lactose, sucrose, and other carbohydrates; a source of nitrogen such as ammonium salts, nitrates, or organic nitrogen sources such as peptones, Wheat bran; mineral salts which supply potassium, sodium, magnesium, nitrate, phosphate, sulfate ions; and trace elements, such as iron; vitamins, especially the vitamins of the B-group; and other mold growth promoting substances. It has been found that the presence of large amounts of phosphates increases the yield. Amounts of potassium monophosphate of at least 1% and preferably of 1.2% to 1.5% of the culture medium have proved to be especially effective.

The pH-value of the culture medium at the start of cultivation is about 4.4 to 4.5. As the mold begins to grow, the pH-value of the culture increases to a pH of 5.2 to 5.8. Cultures with a pH higher than 6.2 did not contain substantial amounts of the active agent. Highest yields of fbrinolytic activity are achieved when discontinuing the cultivation as soon as sporulation sets in. Ordinarily a satisfactory yield is achieved within 3 to days depending upon cultivation temperature and other conditions.

Cultivation can be carried out at a temperature between about 22 C. and about 37 C. The preferred cultivation temperature is between about 28 C. and about 32 C. Substantially no activity was found on cultivation below 20 C. and above 40 C. The fbrinolytic agents may be obtained by both surface or submerged cultivation. The inoculum is grown on a solid culture medium preferably on Sabourauds dextrose medium, until abundant sporulation is observed. Such well sporulated inoculum is then transferred to the liquid culture medium mentioned hereinabove. Three to four inoculums of the size of about 3 cm. in diameter each are transferred to a 5 l. bottle, containing 1,000 cc. of said liquid culture medium. Of course, smaller or larger volumes of culture medium may also be used.

Working up of the culture medium in order to concentrate the fbrinolytic agent may be effected in different ways whereby after each step of isolation the fbrinolytic activity of the resulting intermediate solution and products is determined in order to find out whether a specific step actually causes concentration of the active agent.

One method of producing solutions of the iibrinolytic agents according to the present invention follows closely the method described for testing molds for their fibrinolytic activity. According to said process, the liquid culture medium which has a pH between 5.2 and 5.8 is filtered or centrifuged to remove the mycelium. Preferably filtration is effected through Whatman No. 2 paper. Two parts by volume of ethanol precooled, preferably to -20 C., are added to one part by volume of the filtrate precooled to 0-l0 C. The mixture is kept overnight in the refrigerator below 0 C. or is cooled to 20 C. and centrifuged after standing for half an hour. It was found that best results are obtained when using two parts by volume of alcohol whereby the mixture has an alcohol concentration between about 60% and about 65%. Lower alcohol concentrations than about 40% do not cause precipitation. Higher alcohol concentrations, .e. concentrations above 70%, cause a certain decrease in activity. The alcohol precipitate contains the active agent. This method of working up the culture filtrate, however, yields only a product of low fbrinolytic activity with a very high salt content.

A far superior method of isolating the active agent consists in adding tannic acid to the filtrate of the culture. Preferably such an amount of tannic acid is added that the concentration thereof in the ltrate begins with 25 mg. of tannic acid per cc. of culture filtrate and increases with the amount of fbrinolytic agent in the bath. Tannic acid is preferably added in aqueous 10% solution (100 mg. per cc.). Tannic acid precipitation is carried out at about 0 C. The resulting tannic acid precipitate is about 20 times as active as the alcohol precipitate on a dry weight base. Tannic acid, thus, has a greater selective precipitating effect on the fbrinolytic agent than alcohol since only minimum amounts of salts are trapped in the precipitate.

The following Table I shows the effect of varying amounts of tannic acid added to the mold filtrate. In order to carry out precipitation under exactly the same conditions, the pH-value of the filtrate was adjusted to a pH of 5.5 by the addition of 5 N sodium hydroxide solution before the tannic acid was added. It is evident from this table that there is a relatively insignificant difference between the warious amounts of tannic acid used, provided that an amount of about 0.25 mg. of tannic acid per cc. of culture filtrate is used. The yield is given in this table in mg. per 200 cc. of filtrate. The activity is determined according to the test method described hereinafter after min. and is expressed in percent change in the diameter of the clearing as compared with the diameter at zero time of application of the test solution to the plate. The activity/yield coefficient is expressed as the yield multiplied by the activity percent change. The table clearly shows that best results are achieved with amounts of about 0.75 mg. to 1.0 Ing. of tannic acid per cc. of filtrate.

The activity test is carried out as follows: Modied Astrup-Muellertz iibrin plates are prepared by rapidly adding 1 cc. of bovine thrombin solution containing 100 NIH units/cc. to 10 cc. each of an 0.2% and a 1% l solution of bovine or human fibrinogen. After rapidly mixing said solutions, the mixture is poured into Petri dishes. The plates are then heated at 85 C. for 15 minutes. Thereafter a drop of 0.2 cc. of the mold culture filtrate or suspension or solution to be tested are deposited on each plate by means of a pipette. After incubation in a thermostat at 37 C. up to 180 minutes, the degree of lysis is judged by measuring the area of clearing and digestion of the fibrin plate.

TABLE L-YIELD OF FIBRINOLYTIC AGENT IN RELA- TION TO THE AMOUNT OF TANNIC ACID ADDED TO THE CULTURE FILTRATE Activity in percent increase in diameter o clearing Tannic acid over controls Activity yield added, :ng/cc. Yield mg. at time coeicient The tannic acid precipitate is obtained by centrifuging and decanting the supernatant liquid. The precipitate is washed with anhydrous acetone or any other tannic acid solvent to remove the tannic acid. The remaining active precipitate is then dried under vacuum. The resulting precipitates of active agent, whether obtained by tannic acid precipitation or by alcohol precipitation may be further purified in order to remove insoluble reactive material and contaminating soluble salts. Purification is preferably effected by resuspending the precipitate in water, separating the insoluble material, dialyzing the solution against water, lyophilizing the dialyzed solution.

The chemical, physical, and pharmacological properties of the :librinolytic agent according to the present invention are described in detail in said Pat. No. 3,256,157. More particularly the ibrinolytic agent obtained according to said patent from cultures of Aspergillus oryzae B-82i, Aspergillus oryzae B-1273, Aspergillus flavus B-4m, and Absida coerulea D101 is characterized by not substantially affecting other blood and plasma constituents, being soluble in water, insoluble in ether, ethyl acetate, and acetone, the pH-value of its aqueous solution being between 5.2 and 6.7, aqueous solutions of said fibrinolytic agent losing their brinolytic activity on adjusting their pH-value above a pH of 6.5 and below a pH of 3.0 when standing, the dry iibrinolytic agent being inactivated on standing at a temperature of above 75 C., the infrared spectrum of the alcoholsprecipitated tibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, and near 9.4 microns, and the infrared spectrum of the tannic acid-precipitated brinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, near 6.5 microns, and near 9.4 microns, the ultraviolet absorption spectrum of said tannic acidprecipitated ibrinolytic agent showing an absorption at 280 millimicrons, said fibrinolytic agent not being dialyzable against water through a cellulose membrane with an average pore size of 24 angstroms, the agent being substantially non-pyrogenic and non-antigenic, its aqueous solution causing lysis of thrombin-clotted platelet-poor human and bovine plasma and of thrombin-clotted human and bovine fibrinogen plates.

Experiments with dogs in which a venous blood clot was produced, showed that intravenous injection of 0.250 mg. to 1.0 g. of the tannic acid-precipitated agents suspended in saline solutions caused gradual lysis of the clot and reversal of the biochemical changes in the blood clotting mechanisms which accompany the formation and propagation of the blood clot at the level of thrombosis. A very important additional effect of the new agent is its anti-inflammatory effect at the site of the clot. Furthermore, when experimental clot is formed, it will ordinarily continue to propagate, especially if there is an alteration of the endothelial lining. However, under treatment with the fibrinolytic agents according to the present invention, propagation does not continue, even when the dosage is inadequate to cause complete disappearance of the clot. It was also noted that, under treatment, bleeding occurred at the site of incision from small vessels which had previously spontaneously thrombosed without ligatures.

The brinolytic agents according to the present invention are proteolytic enzymes which have a selective activity of dissolving fibrin. Such fbrinolytic agents have proved to be of considerable importance in the dissolution of blood clots which consist of fibrin. They are administered to animals and humans wherever clotted blood, brinous, or purulent accumulations are undesirably present following trauma or infectious processes which are accompanied by ulceration or abscess formation. They are used in the treatment of suppurative surface tissues. They have also proved of value in the treatment of venous thrombosis as well as of arterial occlusions including coronary thrombosis, as Well as antiinflammatory agents. The brinolytic agents according to the present invention have also been used in the treatment of collagen diseases.

Good results were also obtained on healing chronic resistant osteomyelitis. Amputations for gangrene which heretofore frequently were complete failures, were successfully carried out by administration of the librinolytic agent according to the present invention.

The brinolytic agent according to the present invention is preferably administered by intravenous infusion of its aqueous solution so as to neutralize the patients circulatory antibody and inhibitor. It may also be admin` istered by repeated injections whereby the amounts administered are between 2 mg./ kg. and 10 mg./kg. daily.

Good results are obtained by simultaneous administration of small amounts of heparin or of plasmin. Especially addition of the latter agent causes more rapid breakdown of blood clots than when giving the fibrinolytic agent alone. There is apparently a synergistic effect which these two agents exert upon each other.

It has also proved of advantage first to administer trypsin or the like agents to neutralize the inhibitor present in the blood and then to inject the tibrinolytic agent. This has the advantage that the dose of the brinolytic agent can be reduced considerably.

The brinolytic agent according to the present invention has proved to be very useful in the treatment of wounds. Dead tissue is rapidly digested without substantial damage to living tissue. The brinolytic agent is applied to wounds in powder form or in the form of ointments which preferably contain buffering agents to maintain the most effective pH-value. The iibriuolytic agent rapidly decomposes clotted blood, exudates, and necrotic tissue.

As stated above the brinolytic agent according to the present invention is quite effective in the treatment of suppurative surface tissues such as in the treatment of ulcus cruris varicosum, diabetic and arteriosclerotic gangrene, decubitus sores, abscesses, and others.

When healing infected wounds, the ibrinolytic agent may be combined with sulfonamides and/or antibiotics such as penicillin, tetracycline, bacitracin, neomycin, polymyxin, and others.

The following examples serve to illustrate useful compositions containing the fibrinolytic agent according to the present invention without, however, limiting the same thereto.

7 EXAMPLE 1 Solution for intravenous injection:

Fibrinolytic agent- 100 mg. Saline solution-50 cc.

The fibrinolytic agent is dissolved in the saline solution shortly before use to give an injectable solution containing 2 mg. of active librinolytc agent per cc.

EXAMPLE 2 Solution containing heparin for intravenous injection:

Fibrinolytic agent- 50 mg. Heparin sodium-50 mg. Saline solution-50 cc.

EXAMPLE 3 Solution containing plasmin for intravenous infusion:

Fibrinolytic agent- 50 mg. Plasmin (fibrinolysin)-100 mg. 5% aqueous glucose solution-250 cc.

Shortly before use the -brinolytic agent and plasmin are dissolved in the glucose solution. Each cc. of the solution contains 0.2 mg. of brinolytic agent and 0.4 mg. ,A

of plasmin.

EXAMPLE 4 Ointment for debriding burns Ointment base: Percent Polyethylene glycol dilaurate 60 Polyethylene glycol distearate The fatty acid ester sold under the trademark Glyco Wax S 932 by Glyco Products Company, Inc. Petroleum jelly 5 Paraflin 10 The brinolytic agent is thoroughly mixed with the ointment base. l g. of said ointment contains about l0 mg. of the fibrinolytic agent.

EXAMPLE 5 Powder for the treatment of purulent and necrotic wounds:

G. Fibrinolytic agent 10 Talcum 100 The fibrinolytic agent and the talcum are intimately mixed and the mixture is used as dusting powder on wounds.

The following Table II illustrates the effect of the brinolytic agent obtained according to `Example 6 of Patent No. 3,256,157, i.e. of the tannic acid precipitated ibrinolytic agent, on the lysis of preformed clots in dogs. The tests were carried out as follows:

Clots are formed from autologous dog blood on a special framework enclosed within a clotting tube. The frame consists of a length of polyethylene tubing (outer diameter: 0.6096 mm., inside diameter: 0.279 mm.) threaded through perforated sequins and wrapped spirally with cotton thread. This structure provides a surface to which the clot adheres firmly as it forms and retracts. The frame is enclosed within a piece of clean glass tubing which has not been previously in contact wtih blood and is centered within the tube by passing the ends through the base of a soft rubber stopper and a needle hub. The tube, suspended by means of the polyethylene extending through the needle hub, is maintained in an upright position in an incubator at 37 C. in such a way that the frame is kept in a centered and taut position. A 5 ml. sample of blood is obtained by venipuncture and injected immediately through the base of the rubber stopper, filling the glass tube to a point just above the distal sequin. Air pockets will form in the clot if precautions are not taken to prevent entrance to bubbles while injecting the blood. After incubation for three hours, the clot will have retracted sufficiently to permit removal from the clotting tube. The rubber cuff holding the 18 gauge needle hub is removed first, the glass tube is inverted and dislodged from the rubber stopper, the clot is cut away from the stopper at the junction and is placed in physiological saline. A 5 cm. segment of clot is cut off and a 1 cm. length of polyethylene tubing is left to protrude from one end. The segment is blotted by gently rolling on moistened absorbent paper and weighed. Since a standard length of clot is used, any variation in weight is a result of retraction which tends to alter clot width. Clots formed from dog blood usually weigh between 250-350 mg., excluding the frame which weighs between 35-45 mg. Occasionally clots will weigh more or less than this range when extremes of retraction are encountered. The point of l cm. length of a 27 gauge needle is inserted into the polyethylene tubing which protrudes from the clot and the other end of the needle is placed in the end of a 12 cm. length of No. 20 polyethylene tubing (outer diameter: 1.0922 mm., inside diameter: 0.31 mm.). The clot is then inserted into the cannula made from an 8 cm. length of No. 360 polyethylene tubing (outside diameter: 4.82 mm., inside diameter: 3.76 mm.) with a rubber cuff at one end.

Clot insertion and recovery Dogs weighing between 15-25 kg. are best utilized since the veins are of sulcient size to permit insertion of clots with a diameter produced by the above method. The dog, from which blood was drawn earlier to form the clot, is anesthetized with 30 mg./kg. of sodium pentobarbital intravenously and the neck is shaved. The animal is positioned on its side so that, with the head extended, the external jugular vein and the internal and external maxillary veins are clearly discernible. A 3-4 cm. incision is made on a plane with the internal maxillary vein at a point just distal to the junction of the maxillary branches. A 3 cm. length of the internal maxillary vein is isolated and two loose ligatures are placed around the segment. Pressure is applied over the external jugular vein to distend the branches. A primary ligature is tied distally, and a small incision is made in the wall of the distended internal maxillary vein at a point proximal to the bifurcation. The cannula containing the clot is inserted into the vein and the rubber cuff at the end of the cannula is pinched olf when necessary to control the backtlow of blood. The clot is introduced in the external jugular vein for a distance of 5-6 cm. by means of the polyethylene tubing, and, as the cannula is withdrawn, the secondary ligature is tied around the internal maxillary vein. The clot is now held in place within the jugular vein by means of the polyethylene attachment which is anchored securely in the internal maxillary branch. Unobstructed blood flow is maintained through the large external maxillary vein and collateral circulation into the jugular.

Treatment by injection or infusion of the ibrinolytic agent should be initiated immediately following insertion of the clot. Following treatment, clot removal is accomplished 5 hours after administration of the brinolytic agent by ligating a small segment of the jugular vein at the junction of the frame and polyethylene tubing, cutting the vessel wall, and disengaging the frame from the polyethylene tube. Any clot remaining can be placed in saline solution, blotted, and weighed. The clot is completely removed from the frame which is then weighed separately. The diiference between the preand posttreatment weight, corrected for the weight of the frame, provides the actual amount of dissolution which has occurred and can be expressed as percent lysis.

TABLE II Clot dissolution Body Dose, Dog No Weight ing/kg. Percent Mg 17. 3 5. 50 65. 2 116. 2 20. 5. 00 35. 9 133. 2 21. 8 3. 90 19. 0 76. 6 2l. 5 4. 50 33. 0 120. 4 24. 0 3. 90 14. 7 49. 6 18. l 4. 92 51. 2 224. 2 6 17. 6 4. 25 21. 0 72. 7 Mean of treated dogs- 20. 04 4 57 34. 3 113. 3 227 16. 25 28. 7 82. 7 21. 90 14. 5 53. 8 63. 16. 20 24. 8 80. 8 300 16. 20 +4. 3 +15. 0 391;- 18. 60 14. O 38. 2 395 12.10 +2. 0 +8. 4 Meam of controls 16.87 12. 6 38. 7

It is evident that the clotelysing effect of the brinolytic agent according to this invention is about three times as high as that observed in the control animals. The variations in the clot dissolving power of the fibrinolytic agent from one animal to another one are related to the variations in inhibitor levels of lthe individual animals. Thus selection of the proper dosage, when administering the brinolytic agent parenterally, in human and animal therapy, is dependent upon the amount of inhibitor present in the blood of the patient to be treated.

The presence of components capable of inhibiting proteolytic activity, including the natural fibrinolytic activity of plasmin derived from the activation of the precursor, plasminogen, has been known for many years. An effective method of determining the inhibitor level in blood and thus to take into consideration said inhibitor level when determining the proper dosage required to produce the desired degree of fibrinolytic action without administering too low or too high a dose is described hereinafter as follows. This gravimetric method of assay is based upon determination of the dose of brinolytic agent required to elicit 50% dissolution of a preformed clot in a bulfered system. Incorporation of a 50% dilution of serum or plasma into the system has been found to increase dramatically the dose necessary to effect this degree of dissolution. A quantitative basis for the determination of inhibitory properties of a standard dilution of test serum or plasma has been provided by modication of the assay technique. Inherent proteolytic activity, present in the serum or plasma, can also contribute to the dissolution occurring after addition of a fibrinolytic agent. Thus, the serum inhibitor level, as obtained by the application of the gravimetric method, is an expression of the algebraic sum of the specific and non-specific inhibitor effects in equilibrium with serum proteolytic activity.

The individual serum inhibitor level is dened as the dose in ug. of the standard fibrinolytic agent required to effect 50% dissolution of a preformed blood clot in the presence of a 50% dilution of buifered serum or plasma under standard conditions.

Procedure 0.2 ml. of phosphate buifer of the pH 7.4 are pipetted into a series of l0 x 75 mm. microtubes. To each tube there is added 0.2 ml. of fresh serum or plasma, thereby producing a 50% dilution. Into another series of microtubes there is pipetted 0.4 ml. of the phosphate buffer Without serum or plasma. Clots formed from fresh rabbit blood are blotted, weighed, and introduced into each of the tubes. A volume of 0.1 ml. of an aqueous solution of a brinolytic agent in concentrations of 10 ng. through 150 ,ug/ml., is added to the tubes containing the buffer. Into the tubes containing buttered serum or plasma there is pipetted 0.1 m1. of the same aqueous solution of the ibrinolytic agent in concentrations which are sufficient to produce 20% through 80% dissolution of the clot determined by trial. All tubes are incubated for 2 hours at 37 C., after which the remaining clots are thoroughly blotted and reweighed. A curve is plotted on 3 cycle logprobability paper with the doses of the same ibrinolytic agent in bulfer on the log scale and the percent dissolution on the percentage scale. A similar curve is constructed on the same paper utilizing the data obtained following incorporation of the fibrinolytic agent into a system containing serum or plasma. FIG. 1 presents a graphic illustration of the effect of dog serum on the fibrinolytic activity of the iibrinolytic agent. By interpolation, the dose required to elicit 50% clot dissolution in each system can readily be determined. It is evident from the example illustrated in FIG. 1 that the dose of the brinolytic agent necessary for 50% dissolution in the system containing serum is 7.43 times greater than that required in buffer alone. The individual serum inhibitor level for this sample of blood thus is expressed as 29.0 ng.

Clinical tests have conlirmed the fibrinolytic and thrombolytic activity of the brnolytic agent according to the present invention.

Of course, many changes and variations in the concentration and composition of the brinolytic preparations, the amounts of heparin, plasmin, and other therapeutically effective agents, the methods of administering such fibrinolytic preparations, the dosages administered, and the like may be made by those skilled in the art in accordance with the principles set forth herein and in the claims annexed hereto.

We claim:

1. A process of causing lysis of blood clots in veins and arteries of humans and warm-blooded animals which process comprises intravenously administering into the blood stream of humans or warm-blooded animals an aqueous solution of a brinolytically effective amount of the brinolytic agent obtained from mold cultures selected from the group consisting of Aspergillus oryzae B 82i, Aspergillus oryzae B-l273, Aspergillus flavus B-4m, and Absidia coerulea D-lOl, said agent not substantially affecting other blood and plasma constituents, being soluble in water, insoluble in ether, ethyl acetate, and acetone, the pH-value of its aqueous solution being between 5.2 and 6.1, aqueous solutions of said ibrinolytic agent losing their brnolytic activity on adjusting their pH-value above a pH of 6.5 and below a pH of 3.0 when standing, the dry brinolytic agent being inactivated on standing at a temperature of above 75 C., the infrared spectrum of the alcohol-precipitated brinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, and near 9.4 microns, and the infrared spectrum of the tannic acid precipitated fibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, near 6.5 microns, and near 9.4 microns, the ultraviolet absorption spectrum of said tannic acid-precipitated ibrinolytic agent showing an absorption at 280 millimicrons, the brinolytic agent not being dialyzable against water through a cellulose membrane with an average pore size of 24 angstroms, the agent being substantially non-pyrogenic and non-antigenic, its aqueous solution causing lysis of thrombin-clotted platelet-poor human and bovine plasma and of thrombinclotted human and bovine fibrinogen plates.

2. A method of accelerating healing of purulent and necrotic wounds of humans and Warm-blooded animals which method comprises applying to such wounds a brinolytically effective amount of the fibrinolytic agent obtained from mold cultures selected from the group consisting of Aspergillus oryzae B-28i, Aspergillus oryzae B1273, Aspergillus flavus B-4m, and Absdia coerulea D-lOl, said agent not substantially affecting other blood and plasma constituents, being soluble in Water, insoluble in ether, ethyl acetate, and acetone, the pH-value of its aqueous solution being between 5.2 and 6.1, aqueous solutions of said brinolytic agent losing then flbrinolytic activity on adjusting their pH-value above a pH of 6.5 and below a pH of 3.0 when standing, the dry brinolytic agent being inactivated on standing at a temperature of above 75 C., the infrared spectrum of the alcohol-precipitated brinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, and near 9.4 microns, and the infrared spectrum of the tannic acid-precipitated fibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, near 6.5 microns, and near 9.4 microns, the ultraviolet absorption spectrum of said tannic acid precipitated fibrinolytic agent showing an absorption at 280 millimicrons, the brinolytic agent not being dialyzable against water through a cellulose membrane with an average pore size of 24 angstroms, the agent being substantially non-pyrogenic and non-antigenic, its aqueous solution causing lysis of thrombin-clotted platelet-poor human and bovine plasma and of thrombin-clotted human and bovine fibrinogen plates.

3. An injectable solution for the treatment and dissolution of lblood clots in humans and warm-blooded animals, said solution containing, in aqueous solution suitable for injection, a fbrinolytically effective amount of the fibrinolytic agent obtained from mold cultures selected from the group consisting of Aspergillus oryzae B-82i, Aspergillus olyzae B--l273, Aspergillus flavus B-4m, and Absidia coerulea D-101, said agent not substantially affecting other blood and plasma constituents, being soluble in water, insoluble in ether, ethyl acetate, and acetone, the pH-value of its aqueous solution being between 5.2 and 6.1, aqueous solutions of said fbrinolytic agent losing their fibrinolytic activity on adjusting their pH-value above a pH of 6.5 and below a pH of 3.0 when standing, the dry fbrinolytic agent being inactivated on standing at a temperature of above 75 C., the infrared spectrum of the alcohol-precipitated fibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns7 and near 9.4 microns, and the infrared spectrum of the tannic acid precipitated brinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, near 6.5 microns, and near 9.4 microns, the ultraviolet absorption spectrum of said tannic acid-precipitated fibrinolytic agent showing an absorption at 280 millimicrons, the fibrinolytic agent not being dialyzable against water through a cellulose membrane with an average pore size of 24 angstroms, the agent being substantially non-pyrogenic and non-antigenic, its aqueous solution causing lysis of thrombin-clotted platelet-poor human and bovine plasma and of thrombinclotted human and bovine fibrinogen plates, and further containing a fibrinolytically enhancing amount of plasmin.

4. A topically applicable composition for accelerating healing of purulent and necrotic wounds in humans and warm-blooded animals, said compositions containing a topically applicable pharmaceutically acceptable ointment base and, as active component, fibrinolytically effective amount of the fibrinolytic agent obtained from mold cultures selected from the group consisting of Aspergillus oryzae B-82i, Aspergillus Oryzae B-l273, Aspergillus flavus B-4m, and Absidia coerulea D-lOl, said agent not substantially affecting other blood and plasma constituents, being soluble in water, insoluble in ether, ethyl acetate, and acetone, the pH-value of its aqueous solution being between 5.2 and 6.1, aqueous solutions of said brinolytic agent losing their brinolytic activity on adjusting their pH-value above a pH of 6.5 and below a pH of 3.0, when standing, the dry brinolytic agent being inactivated on standing at a temperature of above 75 C., the infrared spectrum of the alcohol-precipitated fibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6,05 microns, and near 9,4 microns,

Fit

and the infrared spectrum of the tannic acid-precipitated brinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, near 6.5 microns, and near 9.4 microns, the ultraviolet absorption spectrum of said tannic acid-precipitated fbrinolytic agent showing an absorption at 280 millimicrons, the fibrinolytic agent not being dialyzable against water through a cellulose membrane with an average pore size of 24 angstroms, the agent being substantially non-pyrogenic and non-antigenic, its aqueous solution causing lysis of thrombin-clotted platelet-poor human and bovine plasma and of thrombin-clotted human and bovine fibrinogen plates, said active compound being homogeneously dispersed in said ointment base.

5. The topically applicable composition for accelerating healing of purulent and necrotic wounds of claim 4, wherein the ointment base is a pharmaceutically acceptable, substantially anhydrous fatty ointment base.

6. A topically applicable composition for accelerating healing of purulent and necrotic wounds in humans and warm-blooded animals, said composition containing a topically applicable pharmaceutically acceptable pulverulent carrier and, as active component, a fibrinolytically effective amount of the fibrinolytic agent obtained from mold cultures selected from the group consisting of Aspergllus oryzae B-82i, Aspergillus oryzae B-1273, Aspergillus flavus B-4m, and Absidia coerulea D-101, said agent not substantially affecting other blood and plasma constituents, being soluble in water, insoluble in ether, ethyl acetate, and acetone, the pH-value of its aqueous solution being between 5.2 and 6.1, aqueous solutions of said fibrinolytic agent losing their fibrinolytic activity on adjusting their pH-value above a pH of 6.5 and below a pH of 3.0, when standing, the dry fibrinolytic agent being inactivated on standing at a temperature of above 75 C., the infrared spectrum of the alcohol-precipitated fibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, and near 9.4 microns, and the infrared spectrum of the tannic acid-precipitated fibrinolytic agent showing absorption bands near 3 microns, near 3.45 microns, near 6.05 microns, near 6.5 microns, and near 9.4 microns, the ultraviolet absorption spectrum of said tannic acid-precipitated brinolytic agent showing an absorption at 280 millimicrons, the fibrinolytic agent not being dialyzable against water through a cellulose membrane with an average pore size of 24 angstroms, the agent being substantially non-pyrogenic and non-antigenic, its aqueous solution causing lysis of thrombinclotted platelet-poor human and bovine plasma and of thrombin-clotted human and bovine fibrinogen plates, said active component being homogeneously dispersed throughout said pulverulent carrier.

References Cited UNITED STATES PATENTS 9/1965 Maxwell 167-73 7/1966 Truant 195-62 OTHER REFERENCES ALBERT T. MEYERS, Primary Examiner A. P. FAGELSON, Assistant Examiner 

